Importance of Per2 in cardiac mitochondrial protection during stress

During myocardial injury, inflammatory mediators and oxidative stress significantly increase to impair cardiac mitochondria. Emerging evidence has highlighted interplays between circadian protein—period 2 (Per2) and mitochondrial metabolism. However, besides circadian rhythm regulation, the direct role of Per2 in mitochondrial performance particularly following acute stress, remains unknown. In this study, we aim to determine the importance of Per2 protein’s regulatory role in mitochondrial function following exposure to inflammatory cytokine TNFα and oxidative stressor H2O2 in human cardiomyocytes. Global warm ischemia (37 °C) significantly impaired complex I activity with concurrently reduced mitochondrial Per2 in adult mouse hearts. TNFα or H2O2 decreased Per2 protein levels and damaged mitochondrial respiratory function in adult mouse cardiomyocytes. Next, mitochondrial membrane potential (\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$\Delta \psi$$\end{document}Δψ M) using JC-1 fluorescence probe and mitochondrial respiration capacity via Seahorse Cell Mito Stress Test were then detected in Per2 or control siRNA transfected AC16 Human Cardiomyocytes (HCM) that were subjected to 2 h-treatment of TNFα (100 ng/ml) or H2O2 (100 μM). After 4 h-treatment, cell death was also measured using Annexin V and propidium iodide apoptosis kit through flow cytometry. We found that knockdown of Per2 enhanced TNFα-induced cell death and TNFα- or H2O2-disrupted \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$\Delta \psi$$\end{document}ΔψM, as well as TNFα- or H2O2-impaired mitochondrial respiration function. In conclusion, Per2 knockdown increases likelihood of cell death and mitochondrial dysfunction in human cardiomyocytes exposed to either TNFα or H2O2, supporting the protective role of Per2 in HCM during stress with a focus on mitochondrial function.


Per2 expression impacts mitochondrial membrane potential (∆ψ M ) in AC16 human cardiomyocytes subjected to TNFα or H 2 O 2
To evaluate the role of Per2 in regulating mitochondrial vulnerability during stress, we then employed siRNA to knockdown Per2 expression in AC16 cells (human cardiomyocyte cell line).Our data showed a dose-dependent decrease in Per2 mRNA transcripts, with 10 pmol of Per2 siRNA resulting in the smallest reduction of Per2 mRNA compared to control levels, whereas 30 pmol of Per2 siRNA treatment resulted in the greatest reduction (Fig. 2B).We selected a dose of 20 pmol Per2 siRNA in this study, as higher doses of siRNA transfection are injurious to cells, but this dose still induced a significant reduction in Per2 mRNA (Fig. 2B).Western blot analysis further confirmed Per2 protein knockdown with 20 pmol of Per2 siRNA usage in AC16 cells (Fig. 2C).Mitochondrial membrane potential (∆ψ M ) is critical for maintaining mitochondrial function and health.We first assessed mitochondrial membrane potential using JC-1 fluorescent probe in TNFα-or H 2 O 2 -stressed AC16 cells with control or Per2 siRNA transfection.JC-1 displays green fluorescence in the monomeric form in the cytosol upon depolarization, whereas it shows red fluorescence in the aggregated form in active mitochondria.Thus, the ratio of red/green fluorescent intensity indicates mitochondrial membrane potential.In addition to live-cell imaging on AC16 cells using a fluorescent microscope (Fig. 3A), we quantified the ratio of red/green fluorescent intensity using a microplate reader.Our data revealed that 2 h treatment of TNFα or H 2 O 2 damaged ∆ψ M in AC16 cells, denoted by the decreased ratio of red/green fluorescent intensity in these cells compared to their vehicle control counterpart (Fig. 3B).Intriguingly, knockdown of Per2 further reduced TNFα-or H 2 O 2 -impaired ∆ψ M in AC16 cells compared to the control siRNA group (Fig. 3C), displaying a likely protective role of Per2 in maintaining mitochondrial function during stress.However, Per2 siRNA alone did not affect ∆ψ M (Fig. S3).

Per2 knockdown increases cell death in AC16 human cardiomyocytes exposed to TNFα
TNFα or H 2 O 2 can induce cell death in a variety of cells and mitochondrial membrane potential is also involved in apoptosis induction.Therefore, we evaluated the role of Per2 in regulating AC16 cell death following TNFα or H 2 O 2 stimulation.After Annexin V and PI staining and analysis of Flow cytometry (Fig. 4A), our data indicated that 4 h treatment of TNFα or H 2 O 2 significantly increased cell death in AC16 cells (Fig. 4B).Furthermore, Per2 knockdown resulted in greater cell death in TNFα-stressed AC16 cells compared to control.
siRNA transfected cells (Fig. 4C), suggesting that Per2 likely plays a role in TNFα-induced cell death signaling in cardiomyocytes.However, Per2 siRNA alone did not impact cell viability (Fig. S4).

The implication of Per2 in mitochondrial respiration function following TNFα or H 2 O 2 stress
Our previous studies have demonstrated mitochondrial functional impairment in cardiomyocytes subjected to TNFα or H 2 O 2 20,22 .In this study, we determined the role of Per2 in mitochondrial metabolic function in AC16 cells upon exposure of TNFα or H 2 O 2 using Seahorse Cell Mito Stress test (Fig. 5A).While decreased Per2 level did not affect mitochondrial respiration function in AC16 cells without stress of TNFα or H 2 O 2 (Fig. S5), Per2 knockdown did significantly decrease mitochondrial maximal respiration (Fig. 5B) and ATP-linked respiration (Fig. 5C) in AC16 cells subjected to 2 h treatment of TNFα or H 2 O 2 .We further studied effects of stress on mitochondrial oxidative phosphorylation (OXPHOS) complex proteins.Western blot data demonstrated that TNFα significantly increased complex V-ATP5A, complex III-UQCRC2 and complex IV-MTCO1 proteins in AC16 cells (Fig. 6A,C), while a trend of augmented levels of ATP5A and MTCO1 was noticed in AC16 cells   TNFα-induced increase in these complex molecules (Fig. 6B,C).

Discussion
Mitochondria are central to heart bioenergetics, as mitochondrial dysregulation is an underlying mechanism of cardiac dysfunction.In this present study, our data clearly demonstrated that: (1) acute stress reduced Per2 levels and concurrently impaired mitochondrial function in the heart mitochondria and adult cardiomyocytes; (2) Increased cell death and decreased �ψ M were observed in human cardiomyocytes treated with TNFα or H 2 O 2 ; (3) Knockdown of Per2 potentiated TNFα-induced cell death and TNFα-or H 2 O 2 -disrupted �ψ M ; and (4) Per2 knockdown worsened TNFα-or H 2 O 2 -induced impairment of mitochondrial respiration function.These findings supported a strong implication of Per2 in mitochondrial regulation to protect cardiomyocytes during stress.Evidence supports the local inflammation and redox imbalance as important primary factors in cardiomyocyte injury and cardiac functional damage [23][24][25] .Acute injury including ischemia induces excessive production of TNFα or H 2 O 2 that leads to cell death (apoptosis and necrosis) in many types of cells.Our current findings confirmed that TNFα and H 2 O 2 significantly increased cell death in human cardiomyocytes.However, it is unknown whether Per2 plays a role in TNFα-or H 2 O 2 -induced cell death in human cardiomyocytes.
In addition to being a circadian regulator protein, emerging research has suggested the involvement of Per2 in the regulation of cell death through several pathways.Per2 has been found to inhibit activation of PI3K/Akt signaling, thus reducing proliferation while promoting apoptosis in human adenocarcinoma cell lines 26 .In the cytosol, Per2 can function as a scaffolding protein to block mTORC1-induced cell proliferation and increase autophagy in the liver during fasting 27 .Our previous work has also shown that reduction of Per2 in H9c2 cells increased apoptosis following cold storage 10 .In this study, Per2 knockdown enhanced TNFα-induced cell death in human cardiomyocytes, suggesting that Per2 plays a role in the regulation of cell death in cardiomyocytes in response to local inflammatory mediators.TNFα binding to its receptors leads to cell death through multiple regulatory pathways including the extrinsic pathway of apoptosis involving mitochondria 19 .However, the interaction of Per2 and TNFα-initiated cell death pathways are not well elucidated and necessitates further study.
Of note, we did not observe the impact of Per2 in preventing cell death of human cardiomyocytes exposed to H 2 O 2 .Since only one time point (4 h treatment of H 2 O 2 ) was studied, we cannot confirm whether the H 2 O 2 treatment period is suitable to detect cell death here.It is evident that Per2 deficiency reduces the tolerance of H9c2 cells to H 2 O 2 following serum shock, with increased cell death in Per2 knockdown H9c2 cells 28 .However, reduction of Per2 did not significantly affect cell death in the H9c2 cells only exposed to serum shock 28 .This suggests that the relationship between Per2 and cell death may vary depending on the different stressed conditions and the balance of various signaling pathways involved.The exact mechanisms through which Per2 impacts cardiomyocyte cell death are a subject of ongoing research.Future investigations are needed to completely understand the underlying mechanistic details of Per2's role in modulating cell viability.
Recent studies have revealed that the clock regulator Per2 influences mitochondrial metabolic modulation [4][5][6] .Per2 regulates mitochondrial oxidative metabolism in mouse C2C12 myoblasts exposed to fatty acid oxidation 3 .During ischemia, Per2 is activated by the hypoxia-inducible factor1a (HIF-1a) pathway, which facilitates induction of glycolytic enzymes to maximize energy production through glycolysis in low O 2 conditions 5 .Increased oxygen consumption and impaired glycolysis are observed in Per2KO mice subjected to myocardial ischemia.Disruption of Per2 affects mitochondrial morphology and glycogen accumulation following myocardial ischemia 5,8 .Our present findings indicate that Per2 knockdown significantly decreased mitochondrial maximal respiration and ATP-linked respiration in human cardiomyocytes exposed to TNFα or H 2 O 2 .However, the precise mechanisms through which Per2 influences mitochondrial metabolic regulation remains unknown.Emerging evidence has indicated that mutation of Per2 is reported to cause a decrease of complex I activity and a higher NADH/NAD + ratio, thus impacting mitochondrial activity 29 .In addition, Per2 binds to complex IV to mediate mitochondrial respiratory function in endothelial cells following hypoxia 11 .Indeed, the Western Blot data establishes dysregulation of complex III-V protein levels in the AC16 cells with Per2 knockdown, suggesting a potential disruption of the mitochondrial electron transport chain.This is a rational explanation of Per2 knockdown impairing mitochondrial respiratory capacity in human cardiomyocytes.
Notably, mitochondrial membrane potential is essential for preserving normal proton gradient, thus maintaining mitochondrial respiratory function.In this study, TNFα or H 2 O 2 significantly reduced ∆ψ M in human cardiomyocytes.Knockdown of Per2 further worsened TNFα-or H 2 O 2 -impaired ∆ψ M compared to control counterparts.This disrupted ∆ψ M might be reason to explain why Per2 is important in regulating mitochondrial metabolic function.In addition, severe decrease in mitochondrial membrane potential results in changes of mitochondrial structure to release cytochrome C, triggering intrinsic apoptosis through the activation of caspases 30 .This may also explain why reduction of Per2 enhances TNFα-induced cell death in cardiomyocytes.
Overexpression of Per2 via adenovirus provides heart protection from ischemia 31 .Daylight or intense light exposure increases cardiac Per2, leading to reduced myocardial damage following ischemia 5,32 .Intense lightupregulated Per2 overexpression also improves maintenance of endothelial barrier function via promotion of metabolic reprogramming of cellular adaptation to myocardial ischemia 11 .Considering the role of Per2 in modulating cell survival and mitochondrial function during stress from our current study and previous publications as discussed earlier, it is therefore logical that overexpression or delivery of Per2 is protective to prevent cell death in cardiomyocytes and ameliorate mitochondrial dysregulation following myocardia injury 5,33 , thus benefiting mitochondrial bioenergetics and cardiac function.We will explore the therapeutic potential of increasing cardiac Per2 to improve mitochondrial function in our future investigations.
In summary, our findings provide the direct evidence to support that Per2 is important to protect mitochondrial function and cell viability in human cardiomyocytes during inflammatory and oxidative stress (Fig. 7).We speculate that delivery of Per2 protein to cardiomyocytes during myocardial injury could serve as a cardioprotective factor, possibly by supporting mitochondrial function and improving cell survival observed in this study.

Animals
All animal studies were conducted in compliance with the Guide for the Care and Use of Laboratory Animals (NIH Pub.No. 85-23, revised 1996).The animal protocol was reviewed and approved by the Institutional Animal Care and Use Committee of Indiana University.Male C57BL/6J mice (9-12 weeks) were purchased from the Jackson Laboratories (Bar Harbor, ME).The animals were acclimated for > 5 days with a standard diet feeding prior to the experiments and maintained on the same light-dark cycle.All animal experiments were performed between 10:00 am and 2:00 pm.
The study is reported in accordance with ARRIVE guidelines.

Global myocardial ischemia in vivo
After deep anesthesia by isoflurane, the mice were placed supine on a heated pad (37 °C) and injected peritoneally with 0.15 ml of heparin (100 U/ml).The chest was then opened, and the diaphragm was dissected to introduce respiratory arrest.The heart was collected 30 min after the heart stopped beating (30 min-warm ischemia) and snap-frozen in liquid nitrogen.Freshly isolated mouse hearts without global myocardial warm ischemia served as control.Mitochondria were obtained by differential centrifugation using mitochondria isolation kit for tissue (ThermoFisher Scientific) according to manufacturer's protocols.The purity of mitochondrial preparations using this method was confirmed in our previous study 20 .

Measurement of mitochondrial complex I activity
Mitochondrial complex I activity was detected using the enzyme activity microplate assay kit (Abcam, Cambridge, MA, ab109721) according to manufacturer's protocols.Isolated heart mitochondrial pellets (10 μg of mitochondrial protein) were resuspended in PBS with 10% detergent provided in the kit.Enzyme activities were analyzed by increased absorbance OD = 450 nm due to the oxidation of NADH to NAD + using a microplate reader (represented as absorbance changes per minute per milligram protein).Each sample was conducted in duplicate.

Adult mouse cardiomyocyte isolation
By using a Langendorff perfusion system, cardiomyocytes were isolated from adult male mouse hearts based on our previous studies 20,22,34 .Briefly, after mice were heparinized and euthanized with isoflurane overdose, the hearts were excised and rapidly placed into ice-cold calcium-free perfusion buffer.Hearts were perfused and digested with collagenase II (1.5 mg/ml) in perfusion buffer containing 50 mM calcium.Isolated cardiomyocytes were then restored sequentially in perfusion buffer with calcium (100, 250, 500, or 1000 mmol/L CaCl 2 ) and seeded into laminin (20 mg/ml)-precoated 6-well plate in cardiomyocyte plating medium.After 2-h cultivation for adherence, cardiomyocytes were treated with vehicle, 10 ng/ml TNF or 50 μM of H 2 O 2 for 2 h and then collected for protein isolation.The doses of TNFα-and H 2 O 2 were selected based on our previous studies 20,22 .The cardiomyocytes were also plated into laminin (20 mg/ml)-precoated XF96 cell culture plates with 1500 cells/ well 22 .After the cultivation for adherence, the cells were used for the Seahorse XF Cell Mito Stress Assay.

Knocking down of Per2 in AC16 human cardiomyocyte cell line
The AC16 human cardiomyocyte cell line was purchased from the Millipore Sigma and cultured in 100 mm cell culture dishes with DMEM/F12 (Sigma, D6434) containing 12.5% FBS (EMD Millipore, ES-009-B), 2 mM l-Glutamine (EMD Millipore, TMS-002-C), and 1X Penicillin-Streptomycin Solution (EMD Millipore, TMS-AB2-C) at 37 °C, 5% CO 2 and 90% humidity, based on the manufacturer's protocols.AC16 cells were plated in 12-well plate at 1.0X10 5 cells/well or in 96-well plate at 1.0X10 4 cells/well.Twenty-four hour later, cells were transfected with human Per2 or control siRNAs (Life Technologies) using Lipofectamine 2000 (Life Technologies).After one day of transfection, normal AC16 growth medium was added.The cells were allowed to incubate for an additional day and then used for experiments.Different doses of Per2 siRNA (10, 20, and 30 pmol) were used to determine their effects on transcript levels of Per2 and the dose of 20 pmol was selected for subsequent trials in this study.All cell culture studies were performed in accordance with our approved protocol by the Institutional Biosafety Committee of Indiana University.

Real-time quantitative PCR
Total RNA was extracted from AC16 cells using Trizol (Life Technology) and used for the first-strand cDNA reverse transcription using Quantum (ThermoFisher Scientific).TaqMan assays were used to determine transcript levels of human Per2 (Fam dye-labeled) and GAPDH (VIC dye-labeled) (ThermoFisher Scientific) by

Figure 3 .Figure 4 .
Figure 3. Per2 expression impacting mitochondrial membrane potential ( �ψ M ) in AC16 human cardiomyocytes subjected to TNFα or H 2 O 2 .(A) Representative images of mitochondrial �ψ M using JC-1 in control (C) or Per2 siRNA-transfected AC16 cells after 2 h treatment of TNFα or H 2 O 2 .Scare bar = 100 μM.(B) TNFα and H 2 O 2 decreased �ψ M in AC16 cells, represented as folds of the control group. (C) Per2 Knockdown potentiated �ψ M reduction in AC16 cells treated with TNFα and H 2 O 2 , represented as folds of the control in C siRNA or Per2 siRNA group, respectively.Red and green fluorescence intensity in each well was obtained using a microplate reader.The red to green fluorescence intensity ratio was analyzed to indicate �ψ M in (B) and (C).Mean ± SEM, *p < 0.05, ***p < 0.001, ****p < 0.0001.Dot represents individual well.

Figure 7 .
Figure 7. Per2 prevents mitochondrial damage in cardiomyocytes exposed to inflammatory and/or oxidative stress during myocardial injury.